Antioxidant, ACE­inhibitory and antimicrobial activity of fermented goat milk: activity and physicochemical property relationship of the peptide components Article 

Accepted Version 

Moreno­Montoro, M., Olalla­Herrera, M., Rufián­Henares, J. Á., Martínez, R. G., Miralles, B., Bergillos, T., Navarro­Alarcón, M. and Jauregi, P. (2017) Antioxidant, ACE­inhibitory and antimicrobial activity of fermented goat milk: activity and physicochemical property relationship of the peptide components. Food & Function, 8 (8). pp. 2783­2791. ISSN 2042­650X doi: https://doi.org/10.1039/c7fo00666g Available at http://centaur.reading.ac.uk/72237/ 

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Food & Function 2

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Antioxidant, ACE-inhibitory and antimicrobial activity of fermented goat milk: activity 4

and physicochemical properties relationship of the peptide components 5

Miriam Moreno-Montoro,a Manuel Olalla-Herrera,a José Ángel Rufián-Henares,a Rafael 6

Giménez Martínez,a Beatriz Miralles,b Triana Bergillos,c Miguel Navarro-Alarcóna,* & Paula 7

Jauregi,c 8

a Department of Nutrition and Food Science, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071, Granada, 9 Spain 10 b Instituto de Investigaciones en Ciencias de la Alimentación (CIAL, CSIC-UAM, CEI-UAM + CSIC) Nicolás Cabrera 9, 28049, 11 Madrid, Spain 12 cDepartment of Food and Nutritional Sciences, The University of Reading, Whiteknights, PO Box 226, Reading, RG6 6AP, United 13 Kingdom 14

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2

Abstract 18

Increasing evidence on goat milk and their derived products health benefits beyond their 19

nutritional value show their potential as functional foods. In this study, goat milks’ fractions were 20

tested for their total antioxidant capacity measured by different methods (ORAC, ABTS, DPPH 21

and FRAP), as well as the angiotensin-I-converting-enzyme inhibitory and antimicrobial (against 22

Escherichia coli and Micrococcus luteus) activities. Different whey fractions (whey; cation 23

exchange membrane permeate, P and retentate, R) of two fermented skimmed goat milks 24

(ultrafiltered goat milk fermented with the classical starter bacteria or with classical starter plus 25

the Lactobacillus plantarum C4 probiotic strain) were assessed. Additionally, P fractions were 26

divided into two sub-fractions after passing them through a 3 kDa cut-off membrane: (a) the 27

permeate with peptides <3 kDa (P<3); (b) and the retentate with peptides and proteins >3 kDa 28

(P>3). No differences in biological activities were observed between the two fermented milks. 29

However, the biological peptides present in the P<3 fraction showed the highest total antioxidant 30

capacity (for the ORAC assay) and angiotensin-I-converting-enzyme inhibitory activities. Those 31

present in the R fraction showed the highest total antioxidant capacity against ABTS•+ and DPPH• 32

radicals. Some antimicrobial activity against E. coli was observed for the fermented milk with the 33

probiotic, which could be due to some peptides released by the probiotic strain. In conclusion, 34

small and non basic bioactive peptides could be responsible of most of angiotensin-I-converting-35

enzyme inhibitory and antioxidant activities. These findings reinforce the potential benefits of the 36

consumption of fermented goat milk in the prevention of cardiovascular diseases associated to 37

oxidative stress and hypertension. 38

39

Keywords: goat milk, antioxidant, antimicrobial, antihypertensive, ultrafiltration, ion exchange 40

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3

Introduction 43

Fermented milks satisfy daily nutritional requirements for several nutrients and exert different 44

health benefits.1 Furthermore, it is an important source of many bacterial strains owing to the 45

appropriate compatibility among some of them.2 Fermented milks contain several probiotic strains, 46

which additionally increase the already known benefits of these dairy products. Milk fermentation 47

by classical starter bacteria (St) (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus 48

salivarius subsp. thermophilus) changes milk properties and increases its digestibility by a 49

decrease in lactose concentration and pH. This process could also release biological active peptides 50

from their inactive forms present in the corresponding sequence of the precursor protein. The 51

specific sequence and length of released peptides depend on two main factors: (a) the precursor 52

protein, which is different in sequence depending on the animal specie and even on the breed;3 (b) 53

the starter bacteria, since the proteolytic system is inherent to each bacteria strain. The healthy 54

benefits of these bioactive peptides may be attributed to their demonstrated antimicrobial, 55

antioxidant, antihypertensive, antithrombotic, immunomodulatory and opioid activities.4 Many of 56

the bioactive peptides have demonstrated to have multi-functional properties. Nevertheless, their 57

specific activity depends on the amino acid composition as well as sequence. In this sense, it is 58

well known that anionic peptides do not affect gram-negative bacteria because of repulsive 59

electrostatic intractions between the negatively charged outer membrane and the anionic peptides.5 60

On the other hand, some cationic peptides have shown antimicrobial effect against gram-negative 61

bacteria. However, not all the positively charged peptides exert antimicrobial activity and the 62

action mechanism of milk-derived antimicrobial peptides remains uncertain.6 In any case, several 63

peptides have been discovered with antimicrobial activity that can find industrial application.6 64

Among the different functions of bioactive peptides, antioxidant properties are very important 65

because high levels of reactive oxygen species (ROS) and free radicals in the organism are 66

associated to several diseases like cancer, diabetes, cardiovascular diseases, arthritis, allergies as 67

4

well as to aging.7 In addition, ROS presence in food causes quality deterioration and shelf life 68

reduction by lipid oxidation.3 It is known that the defense systems of organisms are often not 69

enough to prevent oxidative damage. Some researchers have stated that antioxidant peptides 70

present in the food system play a vital role in the maintenance of antioxidant defense systems in 71

the organism by preventing the formation of free radicals or by scavenging free radicals and 72

reactive oxygen species, and Cheng et al. even recommended their supplementation.7 An 73

increasing number of food protein hydrolysates and peptides have been found to exhibit 74

antioxidant activity, especially in peptides produced from bovine milk casein.3 In vitro 75

measurement of antioxidant activity is key in the evaluation of the antioxidant potential of 76

bioactive peptide-enriched preparations. Due to the complex nature of antioxidants, there is no a 77

single technique to measure the total antioxidant capacity (TAC) of a food system. Therefore, a 78

variety of analytical techniques are employed with this aim, which can roughly be classified into 79

two types namely, the assays based on hydrogen atom transfer (HAT) reactions and those based 80

on electron transfer (ET).8 Then, to study the antioxidant activity of any sample it is necessary to 81

use at least one assay of each type in order to obtain a more complete evaluation of the TAC as the 82

different mechanisms of antioxidant action will be taken into account;9 this is particularly 83

important when multicomponent samples are being evaluated. 84

Most of biologically active peptides generated from milk proteins have demonstrated an 85

angiotensin-I-converting enzyme-inhibitory activity (ACEi).10 This effect leads to a decrease in 86

angiotensin II (potent vasoconstrictor) and a concomitant increase in the bradykinin level, finally 87

yielding an overall reduction in the blood pressure.11 Although the inhibitory capacity of milk 88

derived peptides is lower than that of chemically designed drugs, their production from natural 89

sources could represent a healthier and more natural alternative for chronic treatment, without the 90

side-effects associated to antihypertensive drugs.11 It is known that most publications on ACEi and 91

5

antihypertensive peptides consider peptides obtained from cow milk.4 However, in recent years 92

goat milk proteins have become an important alternative source of ACEi bioactive peptides.12 93

Previous in vitro studies have demonstrated that the probiotic strain L. plantarum C4 has a 94

positive influence in a range of biological functions such as, mineral bioavailability,13 modulation 95

of the intestinal microbiota14 and protective and immunomodulatory capacity in a murine model 96

of yerseniosis.15 Taking into consideration all previous findings, it was hipothesised here that the 97

probiotic strain could also enhance the antioxidant, ACE-inhibitory and antimicrobial activities, 98

in fermented goats’ milks. 99

Only a few studies have focused on the bioactivity of fermented goat milk peptidic fractions. 100

Therefore, the aim of the present study was the evaluation of the biological activities (antimicrobial 101

activity against Escherichia coli and Micrococcus luteus, TAC measured by ORAC, ABTS, DPPH 102

and FRAP methods, and ACEi-activity) of two fermented skimmed goat milks fermented with the 103

classical starter bacteria [StFM] or with classical starter plus the Lactobacillus plantarum C4 104

probiotic strain [St+LPFM]). The use of the probiotic strain L. plantarum C4 on the milk protein 105

concentrates produced by a local breed of goat for the fermentation process was investigated here 106

for the first time in order to produce a milk product with enhanced biological activities. In addition 107

a novel approach was followed for the physicochemical characterisation (size and charge) of the 108

peptides in the fermented milk in relation to their bioactivities. 109

110

Results and discussion 111

Total protein analysis 112

As stated in Table 1 a significantly higher protein concentration was observed in whey and 113

permeate (P) fractions when compared to the retentate (R), which means a large proportion of the 114

peptides produced by the tested fermenting strains were anionic or nonionic. Additionally, the 115

6

fractions of StFM have a higher protein concentration than St+LPFM; that may be due to 116

differences in the fermentation process between St and L. plantarum C4, in particular pH, as a 117

lower pH was recorded for the fermentation with the probiotic (4.25 ± 0.02) vs. StFM (4.39 ± 0.05) 118

which could have led to more protein coagulation and less soluble protein/peptide.16 119

120

Total antioxidant capacity 121

The results obtained for TAC showed a good correlation with protein content (p<0.001; r: 122

ORAC=0.772, ABTS=0.906 and FRAP=0.950), which could be attributed to the activity of 123

peptides present in those fractions. In order to find which of the fractions had the most active 124

peptides the results were also expressed as µmol Trolox equivalents mg of protein-1 (Fig. 1). The 125

most active fractions were different to those identified when expressed as Trolox equivalents mL-126

1, which means that not always the most active peptides were in the most active fractions. 127

The highest TAC of the fermented milk fractions (Fig. 1) was measured by ORAC for the P<3 128

fraction (reaching 2.927 ± 0.043 µmol Trolox equivalents mL-1 in the StFM) . However, according 129

to the other assays, the different milk fractions did not reach 0.4 µmol Trolox equivalents mL-1 130

(Fig. 1) for any of the fermented milks (StFM and St+LPFM). Thus, in the case of the FRAP and 131

ABTS assays, the highest TAC was found for the whey and P fractions. Therefore these results 132

show that fractionation by IEX did not result in increased activity as whey and P samples had 133

similar TAC according to all methods while the retained fraction had lower activity (particularly 134

according to ORAC and FRAP methods). On the other hand the fractionation by size 135

(ultrafiltration) resulted in significant differences in antioxidant capacity (Fig. 1) with an important 136

increase in activity. P<3 kDa fractionation showed higher values according to ORAC, ABTS and 137

DPPH methods, while no significant differences were observed between these fractions in FRAP 138

assay. 139

7

The measured TAC (by ORAC and ABTS assays) for almost all analyzed fractions was 140

significantly higher for StFM than for St+LPFM (Fig. 1). Only the samples from St+LPFM had 141

significantly higher antioxidant capacity in whey fraction according to DPPH assay. The variation 142

in TAC when using the different methods could be attributed to the presence of different peptides 143

that act by different mechanisms. It has been demonstrated that the TAC of dairy products is 144

mainly due to the activity of peptides. Some authors agreed that the main contribution to TAC 145

comes from casein fractions in milk, suggesting that such effect is related to the self-oxidation of 146

caseins’ amino-acid residues as well as their derived peptides. Additionally, they reported that this 147

activity cannot be replaced by free amino acids since it is the primary structure of casein itself who 148

plays a determining role.17 Among the caseins that release antioxidant peptides, β-CN could be 149

preferably degraded by lactic acid bacteria because it is more unstructured and accessible to 150

cleavage, and therefore hydrolyzed to a greater extent.7 On the other hand, β-LG and lactoferrin 151

have been reported as key components for their high scavenging activity, releasing also peptides 152

with this activity.18 The TAC of peptides has been described as remarkably dependent on factors 153

like molecular weight, amino acid composition and sequence.19 Many authors reported that most 154

of milk protein-derived peptides with antioxidant activity have less than 20 amino-acid 155

residues.1,7,11 This is in agreement with our results as the P<3 fraction, with peptides of MW< 156

3000 (up to about 20 amino-acid residues), had the highest TAC (measured by ORAC), reaching 157

more than 1 µmol trolox equivalents mg protein-1 (Fig. 1). Nevertheless, Virtanen et al.,20 reported 158

the contrary, supporting higher scavenging activity against the ABTS•+ radical of peptides with 159

more than 4 kDa. However, we found that the R fraction contained the peptides with significantly 160

highest TAC against ABTS•+ and DPPH• radicals (~ 0.4 µmol trolox equivalents mg protein-1; 161

Fig. 1). These findings agree with the results reported by other researchers,21 who stated that basic 162

peptides had greater capacity to scavenge hydroxyl radical than weak acidic or neutral ones. 163

Few studies have indicated that the radical scavenging activity is strain-specific and that the 164

higher proteolysis is not always associated with higher TAC.20,22 In our study no significant 165

8

differences were observed for P<3 fraction (µmol trolox equivalents mL-1) between StFM and 166

St+LPFM, and for almost any other fraction when results were expressed as µmol trolox 167

equivalents mg of protein-1. Therefore, the putative probiotic strain L. plantarum C4 by itself or 168

by its interaction with St produced no increase in the antioxidant capacity of the fractions. 169

It is known that goat milk has more β-CN than cow milk. In particular, the analyzed fermented 170

goat milks were concentrated in caseins, therefore it was expected to obtain more β-CN derived 171

peptides than from cow fermented milk. Notwithstanding, results were in the range of those 172

reported for whey fractions of cow fermented milks tested against ABTS, ranging from 0.2774 to 173

2.0356 µmol trolox equivalents mL-1.22 However, the whey fraction had higher TAC than those 174

reported for nonfermented milks (0.489 in UHT and 1.078 µmol trolox equivalents mL-1 in 175

pasteurized milk).23 This finding is probably related to the proteolytic activity of the fermenting 176

strains, which were able to release the antioxidant peptides from milk proteins.24 177

On the other hand, StFM and St+LPFM were produced only in 6 h whereas some authors 178

reported that TAC increases with fermentation time up to 24-48 h.7,22 Some studies reported low 179

TAC of the whey fraction, but after fractionation by HPLC, different fractions with higher TAC 180

were obtained.22 Consequently, future research should focus on fractionating and identifying the 181

peptides responsible of the TAC in the whey fraction. 182

Saura-Calixto and Goñi24 reported a total antioxidant daily intake in a typical Spanish diet of 183

3,549 µmol trolox equivalents (ABTS) and 6,014 µmol trolox equivalents (FRAP). Taking into 184

account the whey obtained from a portion of fermented milk sample (200 g), the percentage for 185

which this whey participate in the daily antioxidant intake is 0.75% for the ABTS and 0.50% for 186

the FRAP methods.24 However, the total antioxidant activity of the fermented milk should be 187

higher if we consider the precipitated fraction, with precipitated caseins and bacteria for which an 188

antioxidant activity has also been reported elsewhere.1 189

Finally, the TAC (Trolox equivalents mL-1) values of the fractions obtained by the different 190

methods were significantly (p < 0.001) correlated with each other (r> 0.830 and r= 0.770 for the 191

9

ABTS-FRAP and ORAC-FRAP, respectively). DPPH was not significantly correlated with any of 192

the other methods. However, when the TAC was expressed based on protein content a significant 193

correlation was also found for DPPH-ABTS (r= 0.937 at p < 0.001) and ORAC-FRAP (r= 0.807 194

at p < 0.001). This additional significant correlation between DPPH-ABTS could be explained by 195

considering mainly the peptides/proteins responsible for the antioxidant capacity. This is very 196

interesting as there was very good correlation between methods testing antioxidant capacity based 197

on the same mechanism, as DPPH and ABTS are based on both hydrogen atom transfer (HAT) 198

and single electron transfer reactions (SET); the highest TAC was found in the retentate according 199

to the ABTS and DPPH methods. Moreover there was also good correlation between methods 200

based on different mechanisms FRAP (SET) and ORAC (HAT) but with biological relevance 201

; the highest TAC was found in permeate according to the FRAP and ORAC methods. These 202

results demonstrate that different types of antioxidants are recovered in the different fractions with 203

differences in their antioxidant mechanism. 204

205

ACEi% activity 206

Firstly, the measured IC50 obtained for captopril was 0.023 µM, in the range reported by the 207

manufacturer (0.021 ± 0.013 μM). This result confirms the reliability of the method used. In Fig. 208

2a, the ACEi activities of the different fractions of fermented goat milks expressed as percentage 209

of inhibition are shown. The whey and P<3 fractions had the highest ACEi activity (about 50%). 210

Interestingly the R fraction did not show any activity. 211

212

Given that in previous in vitro studies13-15 the fermentation by the probiotic strain L. plantarum 213

C4 had led to a range of biological functions the ACEi activity was tested here. Nevertheless, no 214

significant differences were found between StFM and St+LPFM for any of the analysed fractions. 215

Therefore, adding the L. plantarum C4 probiotic strain did not significantly increase the ACEi 216

10

when compared to StFM. Gonzalez-Gonzalez et al.27 found a strain of L. plantarum able to produce 217

a supernatant with high ACEi activity after 24 h of fermentation. Regarding the other 218

microorganisms used, L. bulgaricus has been reported as one of the most proteolytic 219

microorganism as well as a great producer of ACEi peptides25; high ACEi activity (more than 220

50%) was measured in supernatants obtained from milk fermented with 4 strains of L. bulgaricus 221

26. As stated above for TAC, ACEi activity was significantly correlated with protein concentration 222

(r2= 0.800; p < 0.001). When results were expressed as ACEi% mg protein-1, the permeate fractions 223

had the highest activity and in particular the P <3 fraction (Fig. 2b). Therefore, as expected, smaller 224

peptides had the highest ACEi (Fig. 2b). In that sense, the fractionation by size led to an increase 225

in the activity. Interestingly charge had also an effect on activity28 as the positively charged 226

fraction of peptides (R) had very little activity (Fig. 2b). Hence the basic peptides had much less 227

activity than the acidic (negatively charged and noncharged) peptides. This is in accordance with 228

the results of Welderufael et al.,28 who found that one of the fractions of the enzymatic whey 229

hydrolysate with peptides derived from -lactoglobulin with highest ACEi and lowest IC50, 230

contained as main peptides acidic peptides such as IIAE with isoelectric point 4.6. 231

ACEi% reported values for fermented milk whey are very variable depending on the strain 232

used. For milks fermented with L. bulgaricus and S. thermophilus, most of the reported values are 233

around the 50%, ranging from 25% to 70% of ACEi% activity11,25 . Some work was carried out 234

with 13 strains at 3 different final pH’s and found that the maximum inhibitory activity was 51% 235

for milk fermented with Lactococcus lactis 3906 and with final pH 4.3. However, the milk 236

fermented with S. thermophilus did not reach the 18% of ACEi activity.29 Otte et al. demonstrated 237

a negative correlation between pH and ACEi activity of milk fermented with two strains of L. 238

helveticus and two species of the Lactococcus genus, reporting a range from 8 % to 50% of ACEi 239

activity.30 However, higher values of ACEi activity were found in milk fermented with other 240

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strains like Kumis bacteria, ranging from 10.1 to 74.3 % and up to 100% when fermented with St 241

plus L. acidophilus L10, L. casei L26 and B. lactis B9411,31 . 242

On the other hand, the ACEi activity has been demonstrated to be related to ionic calcium 243

(Ca2+), since its concentration may activate or inhibit the ACE.27 We demonstrated that goat UFM 244

was concentrated in caseins and that the ultrafiltration process changed Ca2+ distribution 245

[percentage of Ca associated to caseins changed from 63% in goat raw milk (RM) to 51% in goat 246

UFM] and Ca2+ content from 135.2 ± 10 to 165.6 ± 15.1 mg/100g in goat RM and UFM, 247

respectively. 32 Additionally, the most potent antihypertensive and ACE-inhibitory peptides are 248

generated from caseinates and casein fractions.33 These findings could explain the high ACEi % 249

found in our fermented goat milk samples. Moreover the fermentation with the probiotic L. 250

plantarum did not result in increased ACEi activity. One of its strains was reported to be the best 251

γ-amino butyric acid (GABA) synthesizer; GABA is a non-protein derived amino acid with 252

demonstrated hypotensive effect in rats and humans.34 Future studies should focus on GABA 253

production by the probiotic L. plantarum C4, due to its possible relationship with the hypertension 254

control. 255

256

257

Antimicrobial activity 258

According to the well diffusion assay, no antimicrobial activity of the supernatants against E. 259

coli was observed (p > 0.05). By contrast, in the whey and P fractions, E. coli grew even better 260

than in the control assay. Nevertheless, in the spot assay for both whey and P fractions E.coli did 261

not grow where the drop was placed, probably due to the low pH of the samples (4.33 and 4.59 for 262

whey and P fractions, respectively). However, R fraction, with higher pH (6.97) due to the 263

presence of cationic peptides did not show any activity against E. coli. In relation to M. luteus, we 264

did not find any inhibition neither in the well diffusion assay nor in the spot test. On the contrary, 265

even higher growth was found around the well of the whey fraction compared to the other fractions 266

12

where no effect was shown. Additionally, the co-culture assay was carried out to evaluate more 267

precisely the possible inhibition of E. coli by the studied fractions. None of the fractions of the 268

fermented milk studied showed antimicrobial activity and the pathogen grew almost as much as in 269

the control (Fig. 3). However, after 24 h significant differences in E. coli viable bacteria among 270

control and whey and P fractions of both fermented milks (StFM and St+LPFM), and R fraction 271

of St+LPFM, were found. This inhibition could be due to the acidic pH of whey and P fractions 272

(as mentioned above). However, the R fraction had a pH more similar to the control’s. So in this 273

case, the antimicrobial activity could be due to the cationic peptides isolated in this fraction, such 274

as caprine lactoferricin, which has been shown antibacterial activity against E. coli35. Ionic charge 275

is crucial for the attachment of peptides to the bacterial membrane5; we had hypothesised that 276

cationic peptides would have higher activity than anionic or non charged peptides however, our 277

results did not agree with this. The mechanism of action of milk-derived antimicrobial peptides 278

remains uncertain and other physicochemical properties such as size amphiphilicity and 279

conformation may play a role in their interaction with bacterial membranes. 280

Experimental 281

Samples 282

Goat milk samples from the Murciano-Granadina local breed were obtained from local farms 283

(Granada province, Southeastern Spain). Specifically, every week along five weeks five batches 284

with five samples for StFM and for St+LPFM were done, according to a previously standardised 285

procedure.32 Each individual sample was analysed by triplicate. 286

287

Sample fractionation 288

Fermented milk samples were fractioned in three steps (Fig. 4). In the first step the whey fraction 289

was obtained. All samples were centrifuged at 3000g and 4 ºC for 30 min (Sigma 2-16PK, 290

13

Sartorius, Goettingen, Germany). Then, the supernatant was separated, freeze-dried and stored 291

under refrigeration and nitrogen atmosphere until analysis. Before the fractionation, freeze-dried 292

samples were dissolved in water up to the initial volume and then filtered through 0.22 μm size 293

pore filters Millex® - GS (Merck Millipore Ltd., Cork, Ireland) in a laminar flow cabinet and 294

stored in sterile containers. 295

In the second step a cation exchange was applied. Sartobind filter MA-15 Units (Sartorius, 296

Goettingen, Germany), with a strong acidic cation exchanger membrane. The procedure was 297

carried out according to the operating instructions following four steps: (a) equilibration with 10 298

mL of 10mM potassium phosphate buffer at pH 4.5; (b) loading with 5 mL of sample; (c) washing 299

with 10 mL of equilibration buffer; (d) and finally elution with 5 mL of elution buffer 300

(equilibration buffer + 1 M NaCl at pH 4.5). Then, the cation exchange units were cleaned with 301

0.2 N NaOH for 30 min and equilibrated with 10 mL of equilibration buffer. All steps were 302

conducted at 3 drops/s. With this method, two fractions for each sample were obtained: (1) 303

Permeate (P) composed by anionic or zwitterions peptides and proteins at pH 4.5 that permeates 304

when loading the sample; (2) and Retentate (R) composed by cationic peptides and proteins at pH 305

4.5 retained in the resin and extracted in the elution step. We will refer to them as peptides because 306

we assume that both fractions (P and R) could have bioactivity. 307

In the third step ultrafiltration was applied; molecules will be separated according to size only 308

by a membrane with molecular weight cut off (MWCO) of 3 KDa. (Vivaspin20, Sartorius, 309

Goettingen, Germany), The ion exchange permeates were fractionated into: (1) Permeate ( P<3 ) 310

which contained compounds sized less than 3 kDa anionic or zwitterions peptides; (2) and retentate 311

(P>3) which contained compounds sized more than 3 kDa anionic or zwitterions peptides and 312

proteins. As stated above, we will refer to them as peptides. 313

314

Total soluble protein content 315

14

The total protein content of the samples was determined based on the bicinchonic acid (BCA) 316

assay according to the previously optimized method.36 The absorbance was measured at 562 nm 317

within 10 min using an Ultrospec 1100 pro UV/Visible spectrophotometer (Amersham 318

Biosciences, Little Chalfont, UK). Serial dilutions of bovine serum albumin (Sigma-Aldrich, 319

Steinheim, Germany) were used as standard and bidistilled water as blank. 320

321

Total antioxidant capacity (TAC) measured by ORAC, ABTS, DPPH and FRAP assays 322

The TAC using the oxygen radical antioxidant capacity assay (ORAC) was determined according 323

to the method described by Huang et al.37 slightly modified. In the ABTS assay, the antioxidant 324

capacity was estimated in terms of radical scavenging activity following the procedure described 325

by Pellegrini et al.38 In the DPPH assay, the antiradical activity of different samples was estimated 326

according to the procedure reported by Brand-Williams et al.,39 which was adapted to a microplate 327

reader. Finally for the FRAP determination the ferric reducing ability of each sample solution was 328

estimated according to the procedure described by Benzie and Strain40 and also adapted to a 329

microplate reader. 330

331

332

333

334

335

Measurement of the ACEi% activity 336

The ACE-inhibitory activity of the samples and fractions was measured following the HPLC-based 337

method described by Gonzalez-Gonzales et al.,27 with some modifications. For this aim the 338

determination was done by RP-UHPLC, using a Thermo Scientific Accela UHPLC system (Santa 339

Clara, USA) with thermostated compartment sample injector at 10 ºC and a C18 analytical column 340

(Extrasyl-ODS2, 250 x 4.0 mm, 5 mm, Tecknokroma, Barcelona, Spain) thermostated at 37 ºC. 341

The injection volume was 10 µL and the photodiode array detector was set at 228 nm. The flow 342

15

rate was 1 mL/min with an isocratic solution of acetonitrile 12.5% and trifluoroacetic acid 0.1% 343

in milli-Q water over 8 min, as it was previously reported.41 344

345

Evaluation of the antimicrobial activity 346

This activity was studied using two bacterial strains: a Gram-negative, Escherichia coli K-12 (E. 347

coli), and a Gram-positive, Micrococcus luteus (M. luteus). Before the assay all samples were 348

filtered through 0.22 μm size pore filters (Millex® - GS, Merck Millipore Ltd., Cork, Ireland) 349

under laminar flow and stored in sterile containers. Every measurement was done in triplicate and 350

sterile Phosphate Buffered Saline (PBS, Sigma-Aldrich, Steinheim, Germany) was assayed as 351

blank. 352

The antimicrobial activity of the whey, P and R fractions of StFM and St+LPFM was assayed 353

by the well diffusion assay, based on the method described by Leon Ruiz et al.9 The antimicrobial 354

activity was also evaluated by the spot assay of antibiosis, which was carried out according to the 355

method described by Mohankumar and Murugalatha42 slightly modified. The agar was inoculated 356

with the bacteria prepared as described above. Instead of doing wells, three 20 μL drops of each 357

sample were put on the agar and the plates were incubated as described above. Inhibition zones 358

were measured from the edge of the drop. 359

Finally, for the determination of the antimicrobial activity by the co-culture assay, 4.5 mL of 360

broth culture (NB for E. coli and TSB for M. luteus), 0.5 mL of the sample and 50 μL of the 361

bacteria suspension (growth in NB or TSB at ~ 6-8x108cfu mL-1), were cultured all together. This 362

mixture was incubated under stirring at 37 ºC for E. coli and 30 ºC for M. luteus. Aliquots at t= 0, 363

2, 4, 8 and 24 h were taken, plated out and incubated 24h at 37ºC in NA for E.coli and 48-72 h at 364

30 ºC in TSA for M. luteus. Finally, the colonies were counted and the mean for each plate was 365

calculated and expressed as cfu mL-1. 366

16

367

Statistical analysis 368

The homogeneity of variances was first assessed using the Levene’s test at a significance level of 369

5% (p < 0.05). The data normal distribution was assayed with the Shapiro-Wilk test at a 370

significance level of 5% (p < 0.05). Statistical analysis of data corresponding to different fractions 371

of the same milk type was tested using the ANOVA test when the parametric conditions were 372

fulfilled or using the Kruskall-Wallis test for non-parametric ones. Additionally, to check the 373

existence of statistical differences between same fractions (and whey samples) from different 374

fermented milks (with and without the probiotic) the pair wise independent t-test was used. The 375

evaluation of the relationship between different assays was carried out by computing the relevant 376

correlation coefficient at the p < 0.05 confidence level by Pearson linear correlation (for normal 377

distribution of data) or Spearman linear correlation (for non-normal distribution of data). Analyses 378

were performed using SPSS 17.0 program (Windows version; SPSS Inc., Chicago, IL). The 379

significance value p < 0.05 showed the existence of significant differences. 380

381

Conclusions 382

A remarkable TAC and high ACEi activity for both fermented goat milks (StFM and St+LPFM) 383

were found. The whey was in general one of the most active fractions in all the assays. 384

However the fractionation of the whey according to size and charge gave a very good insight into 385

the relationship between these physicochemical properties (hence chemical structure) and activity 386

measured as antioxidant, antimicrobial and ACEi activity. Interestingly the highest TAC measured 387

by ORAC was found in the P<3 fraction, therefore peptides with MW<3000 Da were the main 388

contributors to the antioxidant activity not the proteins. On the other hand, positively charged basic 389

peptides (those in the retentate fraction of the membrane separation step) had the highest TAC 390

against ABTS•+ and DPPH• radicals; both methods test antioxidant mechanism according to HAT 391

17

and SET mechanisms. In terms of ACEi activity, the highest activity was found in the P<3 fraction. 392

So the smallest (nonionic and anionic) peptides were the main contributors to the ACEi and 393

antioxidant (according to ORAC) activities of the whey. 394

None of the samples had antimicrobial activity against the gram positive bacteria. The whey and 395

the anionic/nonionic fractions of the fermented milk with the starter had some antimicrobial 396

activity against the gram negative bacteria however, this may be partly due to the low pH. Only 397

the whey and the cationic fraction of the fermented milk with the probiotic showed some activity 398

against E.coli which could be attributed to peptides released by L. plantarum C4 during the 399

fermentation process such as those derived from lactoferrin. 400

Finally, the activities attributed to the whey fractions show potential health benefits of the 401

consumption of fermented goat milk. However, further research is needed to conduct clinical trials 402

to substantiate these and for further identification of individual peptides responsible for the 403

activities. 404

405

Acknowledgements 406

407

This study was supported by the project P09-AGR-4195 from the Regional Ministry of 408

Innovation, Science and Business (Andalusia). The authors would like to thank the Spanish 409

Ministry of Education for a grant awarded to M. Moreno-Montoro (Ref. AP2009-3138) and to 410

the Spanish Ministry of Economy and Competitiveness (MINECO) for the project AGL2015-411

66886R 412

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465

466

467

468

20

469

470 Table 1. Total protein content in the different fractions of goat fermented

milks (mean ± SD, mg mL-1)

Sample

type n Whey fraction P fraction R fraction

P<3 KDa

fraction

P>3 KDa

fraction

StFM 25 6.78±0.773* 5.69±0.548# 0.436±0.096 2.23±0.145 1.31±0.377

St+LPFM 25 5.70±0.661* 4.30±0.843# 0.355±0.055 2.08±0.127 0.97±0.142

Mean

value 50 6.16±0.868a,* 4.85±0.990b,# 0.388±0.076c,** 2.14±0.143d,## 1.19±0.225e,

StFM: Fermented milk manufactured with skimmed milk concentrated by ultrafiltration (UFM) and fermented with the classical starter bacteria (St: L. bulgaricus and S. thermophilus); St+LPFM: Probiotic fermented goat milk manufactured with

UFM and fermented with St and L. plantarum C4; Whey fraction: Fermented milk supernatant after centrifugation; P fraction:

IEX (Ion exchange) permeate; R fraction: IEX retentate; P<3 fraction: P fraction with less than 3 kDa molecular weight; P>3 kDa fraction: P fraction with more than 3 kDa molecular weight.

*,#Statistical differences between the same fractions of StFM and St+LPFM: p < 0.05.

a,b,c,d,e,Superscripts with different letters indicate the existence of statistical differences among different fractions: *p < 0.01;

**,##,p < 0.001).

21

Table 2. Final pH of the co-culture supernatants at 24h for fermented goat milks (StFM

and St+LPFM) and control

Sample n Whey fraction P fraction R fraction Control

StFM (TSB) 25 5.04 ± 0.07 5.06 ± 0.01 7.46 ± 0.07 7.30 ± 0.18

St+LPFM (NB) 25 4.91 ± 0.07 4.83 ± 0.01 6.64 ± 0.01 6.85 ± 0.12

The pH was measured in the supernatant of the culture media mixed with the fractions after the assay. TSB: Tryptone soy broth culture media; NB: Nutrition broth culture media; WHEY: Fermented milk supernatant after centrifugation; P: IEX (Ion exchange) permeate; R: IEX retentate;

Control: Sterile PBS.

471

472

22

473

ORAC

(1)

ABTS

DPPH

FRAP

Fig. 1. Antioxidant activity (TEAC mL-1 and TEAC mg protein-1) of the fermented milk fractions

by ORAC, ABTS, DPPH and FRAP assays

StFM: Fermented goat milk manufactured with skimmed goat milk concentrated by ultrafiltration (UFM) and fermented with the classical

starter bacteria (St) L. bulgaricus and S. thermophilus; St+LPFM: Probiotic fermented goat milk manufactured with UFM and fermented

St and L. plantarum C4; Whey fraction: Fermented milk supernatant after centrifugation; P fraction: IEX (Ion exchange) permeate; R

fraction: IEX retentate; P<3 fraction: P fraction with less than 3 kDa molecular weight; P>3 fraction: P fraction with more than 3 kDa

molecular weight.

*,#,,**,##,,,***,###,Statistical differences between values for StFM and St+LPFM: *,#,p < 0.05; **,##,,p< 0.01; ***,###,p< 0.001 a,b,c,d,eSuperscripts with different letters indicate the existence of significant differences among fractions (letter : p < 0.05; letter,*,#,: p < 0.01; letter, **,##,,: p < 0.001).

474

a

***b

###

c **

d*

e #

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

L

StFM St+LPFM

a*b**

#

c

d##

e

0.0

0.5

1.0

1.5

2.0

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

g p

rote

in

StFM St+LPFM

a

***a

###

b**##

c

d

*

0.0

0.1

0.2

0.3

0.4

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

L

a*b**

#

c##

d

b

0.0

0.1

0.2

0.3

0.4

0.5

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

g p

rote

in

ab

* ab

c**

d#

0.0

0.1

0.2

0.3

0.4

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

L

a

**b

c##

d*ab

0.0

0.1

0.2

0.3

0.4

0.5

0.6

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

g p

rote

in

a**a##

*

b**

c c

0.00

0.10

0.20

0.30

0.40

0.50

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

L

ab

c*

b#b

0.00

0.02

0.04

0.06

0.08

WHEY P R P<3 P>3

μm

ol

TE

AC

/ m

g p

rote

in

23

475

476

477 478

479

Fig. 2. Angiogensin-I-converting-enzyme inhibitory activity (ACEi) of StFM and St+LPFM 480 expressed as percentage of ACE inhibition (a) and inhibitory efficiency ratio (IER; b). 481

StFM: Fermented goat milk manufactured with skimmed goat milk concentrated by ultrafiltration (UFM) and fermented with the 482 classical starter bacteria (St) L. bulgaricus and S. thermophilus; St+LPFM: Probiotic fermented goat milk manufactured with 483 UFM and fermented St and L. plantarum C4; Whey fraction: Fermented milk supernatant after centrifugation; P fraction: IEX 484 (Ion exchange) permeate; R fraction: IEX retentate; P<3 fraction: P fraction with less than 3 kDa molecular weight; P>3 fraction: 485 P fraction with more than 3 kDa molecular weight. 486 *,#,,**,##Statistical differences between values for StFM and St+LPFM: *,#,p < 0.05; **,##p< 0.01 487 a,b,c,d,eSuperscripts with different letters indicate the existence of significant differences among fractions (letter : p < 0.05; letter, *,#,: 488 p < 0.01; letter,**,##: p < 0.001). 489

490 491

492

a*

b#

c**

a

b##

0

10

20

30

40

50

60

WHEY P R P<3 P>3

AC

Ei

(%)

(a)StFM St+LPFM

a**d a

b##

c*

d

0

5

10

15

20

25

30

WHEY P R P<3 P>3

% A

CE

/mg p

rote

ína

(IE

R)

(b)

24

493

494

Fig. 3. Antimicrobial activity measured as viable E. coli after co-culture with the different 495 fractions from StFM (a) and St+LPFM (b) 496

StFM: Fermented goat milk manufactured with skimmed goat milk concentrated by ultrafiltration (UFM) and fermented with the 497 classical starter bacteria (St) L. bulgaricus and S. thermophilus; St+LPFM: Probiotic fermented goat milk manufactured with UFM 498 and fermented St and L. plantarum C4; Whey fraction: Fermented milk supernatant after centrifugation; P fraction: IEX (Ion 499 exchange) permeate; R fraction: IEX retentate; P<3 fraction: P fraction with less than 3 kDa molecular weight; P>3 fraction: P 500 fraction with more than 3 kDa molecular weight; Control: sterile PBS. 501 *,***,###Significant differences for viable E. coli at specific time among fractions of fermented goat milks and the control: *p < 502 0.05; ***,###p< 0.001. 503 504

505

506

507

508

509

510

511

512

513

WHEY***

WHEY *WHEY & P

###

4

5

6

7

8

9

10

0 5 10 15 20 25

log C

FU

/mL

Time (h)

(a)WHEY P R Control

WHEY & P

***

R*

R & P

WHEY, P &

R###

4

5

6

7

8

9

10

0 5 10 15 20 25

log C

FU

/mL

Time (h)

(b)

25

514 515

516 517 518

519 520 521 522 523

524 525 526 527 528

529 530 531

532 533 534 535

536

Fig. 4. Sample fractionation diagram for skimmed goat milks with classical starter bacteria 537 (StFM) and with the classical starter St plus Lactobacillus plantarum C4 probiotic strain 538

(St+LPFM) 539 Whey: Fermented milk supernatant after centrifugation; Cationic fraction: Ion exchange (IEX) permeate; Anionic fraction: IEX retentate; P<3 540 fraction: P fraction with less than 3kDa molecular weight; P>3 fraction: P fraction with more than 3kDa molecular weight. 541 542

543

544

545

546

Goat fermented milk

StFM

St+LPFM

1. Centrifugation

Whey fraction

2. Ion Exchange

Anionic

fraction

3. Ultrafiltration (3 kDa)

Cationic

fraction

P < 3 KDa

P > 3 KDa

26

547

548

549

550

551

552

Graphical abstract 553

554

555